The purpose of this experiment consisted of the proper isolation and identification of possible novel marine microorganisms using 16S rRNA gene phylogeny and an array of biochemical identification tests. The successful isolation of novel species, or newly discovered species, has become an important goal for many researchers, especially recently. Perhaps the biggest motivation for the isolation of these new species is antibiotics. The continued rise in antibiotic resistance has lead to a shortage of effective antimicrobial products as well as a deficit in organisms that can provide these necessary products for the production of semi-synthetic antibiotics (Dalisay 2013). Being that the majority of marine habitats still remain uncharted and unexplored, beaches and oceans have become a great focus of recent research efforts in discovering novel species (Delisay 2013). Furthermore, the great diversity of organisms found in these habitats makes marine environments very desirable scientific research territory.
The most important aspect of novel marine organism identification is the 16S rRNA gene. This specific gene is found in all prokaryotes and serves as a distinguishing factor between bacterial species (Janda and Abbott 2007). What makes 16S rRNA a desirable gene in prokaryote identification is the stability and consistency of its function over the years, thus resulting in decreased identification errors as well as reduced sequence changes (Janda and Abbott 2007). Additionally, the size of this gene, consisting of about 1.5 kb, is large enough for processing phylogenetic information but small enough for relatively rapid sequencing and analysis (Clarridge 2004). Furthermore, 16S rRNA can aid in the construction of phylogenetic trees, which can be helpful in depicting relationships between different bacterial species as well as finding close relatives of specific novel organisms. In addition to 16S rRNA analysis, biochemical characterization tests were another crucial aspect of novel marine bacteria identification.
Biochemical tests can be helpful in understanding fermentation, hemolysis, pathogenicity, and other biological aspects of a microorganism. Agar plates along with agar slant tests can provide great insight into bacterial metabolism. MacConkey (MAC) agar plates, for example, are selective media that favor the growth of Gram-negative bacteria. Additionally, MAC agar plates are used to differentiate between lactose fermenting and non-lactose fermenting microorganisms (John 2016). Lactose fermentation in MAC plates is noted by a color change in the agar from pink to yellow. Mannitol salt agar plates, or MSA plates, are another popular biochemical characterization test. These agar plates are very selective for the growth of Gram-positive halophiles, specifically, those belonging to the genus Staphylococcus (John 2016). Furthermore, mannitol fermenters are characterized by the active color change of the phenol red agar to yellow.
Blood agar plates are useful in identifying the hemolytic aspects and relating these hemolytic properties to the pathogenicity of an organism (John 2016). Other tests, such as Triple Sugar Iron (TSI) test, are also used to gain a better understanding of the metabolism of specific microorganisms. These biochemical tests along with 16S rRNA analysis, PCR cleanup, and gel electrophoresis can aid in the proper identification of novel marine species.